This invention relates to the use of small molecules for modulating the functions of transcription factors that are associated with certain diseases. Transcription factors are proteins that bind to specific DNA sequences and regulate gene expression either directly or through associated proteins such as co-activators and co-repressors, or by recruiting histone modifying enzymes such as histone acetyltransferases (HATs) and histone deacetylases (HDACs). Transcription factors play key roles in many biological processes by ensuring the appropriate level of gene expression. They can also be associated with certain disease states, if their ability to regulate transcription is aberrantly modified. The identification and development of small molecules that can selectively modulate the function of certain transcription factors, therefore, can lead to potentially new therapeutic applications. This invention is based on two basic ideas. One is to develop small molecules that bind a specific transcription factors such as MEF2, FOXP3 and GATA3 and modulate its interaction with transcription co-activators and co-repressors. The other is to develop small molecules to block the recruitment of HAT (such as p300 and CBP) and HDACs and other histone modifying enzymes (such as histone methyltransferases, and demethylases, DNA methyltransferases) and chromatin remodeling machineries to specific regions of chromatin.
In particular, this invention relates to the myocyte enhancer factor-2 (MEF2) which plays critical roles in the development and adaptive responses of the muscle, immune and nervous systems (Flavell et al., 2006; Kim et al., 2008; Mao et al., 1999; McKinsey et al., 2002; Pan et al., 2004; Potthoff and Olson, 2007; Youn and Liu, 2000; Youn et al., 1999). MEF2 has been implicated as a key regulator of hypertrophic responses in heart muscle cells. Heart hypertrophy induced by pathological stimuli can lead to heart failure in many forms of cardiovascular diseases.
MEF2 generally defines a family of transcription factors with four members: MEF2A, MEF2B, MEF2C and MEF2D. The importance of their function has been demonstrated in detail through the use of murine and Drosophila genetics (Potthoff and Olson, 2007). MEF2, in skeletal muscles where it was initially identified, together with myogenic basic helix-loop-helix transcription factors such as MyoD, promotes and maintains myogenesis (Molkentin and Olson, 1996). MEF2A, one member of the MEF2 family, has recently been coined as the “heart attack gene” because a mutation in this protein is linked to coronary artery disease (CAD) and myocardial infarction (MI) (Wang et al, 2003). These findings underlie a critical role of MEF2 in human heart diseases (Kim et al., 2008; Wei et al., 2008; Zhang et al., 2002).
MEF2 is now known to be a general transcriptional factor in many other cell types. For instance, MEF2 is one of the important transcriptional factors for mediating calcium signaling in lymph system development} (Pan et al., 2004; Youn and Liu, 2000; Youn et al., 1999), MEF2 regulates cytokine expression and immune responses. MEF2 also regulates transcription programs underlying neuronal survival and synaptic remodeling (Chen and Cepko, 2009; Flavell et al., 2006; Flavell and Greenberg, 2008; Flavell et al., 2008; Mao et al., 1999; Morrow et al., 2008; Shalizi et al., 2006; Shalizi and Bonni, 2005; Yang et al., 2009). These observations suggest that small molecules that modulate MEF2 function could have therapeutic effect in cardiac hypertrophy and heart failure, autoimmune diseases and transplant rejection, neurodegenerative diseases and pathological impairment of learning and memory (Fischer et al., 2007; Stefanko et al., 2009).
Inside cells, the action of MEF2 includes three distinct steps: (i) transcriptional repression; (ii) calcium-dependent de-repression; and (iii) transcription activation. Transcriptional repression by MEF2 depends on its association with a variety of transcriptional co-repressors with intrinsic or associated histone deacetylase (HDAC) activity. In T cells, MEF2 bind Cabin1, which in turn associate with class I HDACs such as HDAC1, HADC2 and HDAC3 (Youn and Liu, 2000). In muscle cells, MEF2 binds directly to class II HDACs such as HDAC4, HDAC5, and HDAC9 and inhibits the expression of specific genes involved in the development and adaptive responses of muscle (Chan et al., 2003; Gregoire et al., 2006; Gregoire and Yang, 2005; McKinsey et al., 2001, 2002; Miska et al., 1999; Sparrow et al., 1999). Mice whose HDAC5 or HDAC9 has been knocked out showed increased sensitivity to hypertrophic stimuli, suggesting important roles of class II HDACs in heart hypertrophy (Potthoff and Olson, 2007). These and other data indicate that MEF2 and Class II histone deacetylases (HDACs), and particularly HDAC5 and HDAC9, are key mediators of hypertrophic signals in cardiomyocytes. Ample data have suggested that the MEF2/class II HDAC pathway is potential therapeutic target for heart hypertrophy.
In response to specific calcium signals, Cabin1 and HDACs are removed from MEF2 (Potthoff and Olson, 2007). MEF2 then recruits co-activators such as p300 and CBP to turn on distinct programs by association with a variety of transcriptional activators and co-activators (McKinsey et al., 2001; Sartorelli et al., 1997; Slepak et al., 2001; Wei et al., 2008). A small increase of p300 has been shown to be necessary and sufficient to induce MEF2-dependent cardiac hypertrophy. MEF2 has a highly conserved N-terminal region (residues 2-93), consisting of the well-characterized MADS-box and a MEF2-specific domain (Shore and Sharrocks, 1995). The MADS-box/MEF2 domain is remarkably rich in function, mediating DNA binding, dimerization, and protein-protein interactions with a myriad of MEF2 transcription partners (McKinsey et al., 2001, 2002), including Cabin1, Class IIa HDACs and p300/CBP. It has been shown that the MADS-box/MEF2 domain in MEF2 is necessary and sufficient to bind with a small motif conserved in class II HDACs and Cabin1. The CH3 domain of p300 and CBP is also shown to bind the MADS-box/MEF2 domain.
Despite the extensive knowledge about MEF2's involvement in various cellular processes available in the art, it has heretofore been impossible to capitalize on the knowledge due the lack of suitable molecular tools. In particular, how to modulate the activity of MEF2 by small molecules has been a long standing challenge. This is because MEF2 is a relatively small transcription factor without any apparent enzymatic activity; its main function is to bind specific DNA and recruit transcription co-regulators such as Cabin1, class II HDACs and p300/CBP to specific promoters. This mode of function is generally considered not druggable or at least very difficult to target by small molecules. Discovery or creation of such molecules will facilitate further advances in this field and can lead to new mechanism-based and structure-based therapeutic applications for MEF2-associated diseases, including inflammation, autoimmune diseases, neurodegenerative diseases, cancer, and cardiovascular disease.
The invention described in this application can also be extended to modulating the activity of other transcription factors such as the forkhead/winged helix transcription factor FOXP3 (Bennett et al., 2001; Fontenot et al., 2003; Hori et al., 2003; Wu et al., 2006; Zheng and Rudensky, 2007). FOXP3 is a key transcription factor critical to the development and function of regulator T cells (Tregs). Tregs are a special population of T cells required for suppressing the excessive activation of the immune system. Loss of function of FOXP3 by mutations and other mechanisms lead to fetal autoimmune diseases such as IPEX whereas enhanced expression of FOXP3 or its activity can confer suppression function. Elevated FOXP3 function can be beneficial in treating autoimmune diseases and transplant rejection while strategic down regulation of FOXP3 activity can be used to develop immune-based anti-tumor therapies (Zuo et al., 2007a; Zuo et al., 2007b). Thus, small molecules that bind FOXP3 and modulate its interaction with co-repressors and co-activators could have therapeutic application in autoimmune diseases, transplant rejection and cancer therapy.
Similarly to MEF2, the function of FOXP3 is tightly regulated by transcription co-regulators that include HAT (such as TIP60) and HDACs (including class I and class II HDACs) (Li et al., 2007). Thus, small molecules could be developed by methods described in this invention that binds FOXP3 and blocks its interaction with co-regulators including epigenetic regulators such as histone modifying enzymes and chromatin remodeling machines.
Similarly, targeting transcription co-regulators has also met its share of challenges.
Among the transcription co-regulators of transcription factors such as MEF2, class II HDACs are the best studied group.
Histone deacetylases (HDACs) (EC number 3.5.1) are a class of enzymes that remove acetyl groups from an ε-N-acetyl lysine amino acid on a histone. Its action is opposite to that of histone acetyltransferases (HATs). HDACs proteins are now also being referred to as lysine deacetylases (KDAC) to more precisely describe their activity rather than their target, which also includes numerous non-histone proteins.
As their name suggests, one of HDACs main functions is to remove acetyl groups from histone proteins. Histone proteins are the chief protein components of chromatin. They act as spools around which DNA winds and play an important role in gene regulation and DNA packaging. Histone proteins have tails that are normally positively charged due to the amine groups present on their lysine and arginine amino acids. These positive charges help the histone tails to interact with and bind to the negatively charged phosphate groups on the DNA backbone.
The association between DNA and histone acts as a vital control mechanism in regulating the ability of transcription factors to access DNA. Strong association between DNA and histones restricts access by transcription factors and therefore represses gene transcription (Morrison et al., 2007). Modification of histones or DNA can alter the strength of their association and thus modulate transcriptional activity (Morrison et al., 2007). Covalent addition of methyl, phosphate, or acetyl moieties has been shown to alter the nucleosome state and consequently affect transcription. Acetylation results from the addition of an acetyl group to the ε-amino group of conserved N-terminal lysine residues on histones. Addition of acetyl groups to histones reduces the attractive force between positively charged histone proteins and the negatively charged DNA phosphate backbone, resulting in a more relaxed and accessible chromatin structure. HATs facilitate histone acetylation and are thus believed to be transcriptional activators. Conversely, HDACs serve to remove acetyl groups from histones and thereby repress transcription. Thus, it is the interplay between HATs and HDACs activity that primarily governs local chromatin structure and gene expression. HDACs alter global gene transcription through the deacetylation of chromatin. It should be noted that HDACs do not directly bind DNA sequence and require additional factors for target gene recognition (Morrison et al., 2007).
There are 4 recognized subtypes of HDAC proteins (class I-IV) based on function and DNA sequence similarities. The first two subtypes are considered “classical” HDACs whose activity are inhibited by trichostatin A (TSA), Class I HDACs includes HDACs 1, 2, 3 and 8, which are expressed ubiquitously (Zhang and Olsen, 2000). Class II HDACs has two subgroups, Ha that includes 4, 5, 7 and 9, and IIb that includes HDAC6 and HDAC10. Class IIa share a common structural organization, with carboxyl-terminal catalytic domain and an amino-terminal extension that mediates interactions with members of the myocyte enhancer factor 2 (MEF2) family of transcription factors. HDACs also differ in terms of their subcellular localization with class I generally found in the nucleus, class IIb are located mostly in the cytoplasm, while class IIa shuttle between the nucleus and the cytoplasm. Unlike class I HDACs, class IIa HDACs are tissue-restricted, with especially high levels of expression in heart, skeletal muscle, and brain (Zhang et al., 2002). Class III is a family of NAD+-dependent proteins not affected by TSA and class IV is considered an atypical category of its own. HDAC11 is grouped in class V.
Given the important roles that HDACs play in cellular processes, the medical applications of HDAC inhibitors (HDACi) is an intense area of research. However, many uses of HDACi in medicine were discovered without knowledge of the underlying mechanism. For example, in psychiatry and neurology, there is a long history of using valproic acid as mood stabilizers and anti-epileptics. The anticonvulsant property of valproic acid was accidentally discovered when it was being used as a vehicle for a number of other compounds that were being investigated as anticonvulsant. It was not until later that valproic acid was identified as a HDACi. In recent years, HDACi are being actively studied as a mitigator or treatment for neurodegenerative diseases. There has also been extensive effort to develop HDACi for cancer therapy. For example, Vorinostat (SAHA) has recently been approved for treatment of cutaneous T-cell lymphoma (CTCL). An alternative agent under clinical evaluation for CTCL is the cyclic depsipeptide natural product FK228 (Romidepsin) which is a potent inhibitor of class I HDACs. In addition, a clinical trial is studying the effects of valproic acid on the latent pools of HIV in infected persons. Despite the growing interest in the medicinal applications of HDACi, the exact mechanisms by which these compounds work are still not well understood. Thus, these efforts are largely guided by guesswork and trial-and-error experiments.
One particular problem with the use of HDACi is that most of the known small molecules that inhibit HDAC activity are designed to function by targeting the catalytic activity of HDACs. However, since the active site is a conserved feature shared by a large number of different HDAC isoforms, it is inherently difficult to identify isoform-selective HDACi. Therefore, most HDACi have low specificity and are incapable of specific targeting of any particular species of HDAC. For example, trichostatin A (TSA) is among the most potent reversible HDACi currently known, with an IC50 in low nanomolar range. TSA with its hydroxamic acid group and its five-carbon atom linker to the phenyl group, has the optimal conformation to fit into the active site of HDAC (de Ruijter et al., 2003; Somoza et al., 2004). All HDACs are thought to be approximately equally sensitive to inhibition by TSA (de Ruijter et al., 2003).
A major impediment, therefore, for the discovery of small molecules that inhibit the function of HDACs and thereby modulate the activity of related transcription factors, is that the current state of the art is focused on the discovery and optimization of HDACi that are identified and evaluated through their ability to bind to the active site of the HDAC enzymes. Typically, these HDACi have the general structure R-L-Z, where R is a protein surface recognition group connected via a short fatty linker L to a Zn2+-chelating group Z that binds to the active site zinc atom. The most common chelating groups (Z) featured in the known HDACi are: hydroxamic acids (ISA, vorinostat, LAQ824, belinostat), thiol derivatives (FK228, largazole) or electrophilic ketones (trapoxin A). A potential drawback of such groups that bind tightly to metal cations like Zn2+ is that they may lack sufficient selectivity for a particular protein, resulting in various side effects.
Another class of known HDACi are the benzamides that feature an ortho-aminoanilide (2-aminoanilide) moiety, including MS-725, MGCD0103, pimeloylanilide ortho-aminoanilide (PAOA) and compound 106 (N1-(2-aminophenyl)-N7-p-tolylheptanediamide) which was investigated as a potential therapeutic for neurodegenerative diseases including Friedreich's ataxia and Huntington disease (Chou et al., 2008; Herman et al., 2006; Paris et al., 2008; Rai et al., 2008; Thomas et al., 2008; Wong et al., 2003). Although the detailed molecular mechanism of action of this class of HDACi is not known, these molecules were postulated to involve binding of the o-aminoanilide group to the zinc atom of the HDAC active site, despite the lack of any direct evidence regarding such binding motif. Moreover, such molecules were found to exhibit biological activity implicating inhibition of HDAC function, even though other non-selective HDACi did not show similar activity. For example compound 106 was shown to be very active for the induction of frataxin, despite weak HDAC inhibition, while closely related HDACi such as SAHA did not have this type of activity. Consequently, in light of the absence of a molecular mechanism for the actions of this class of compounds, the optimization of their therapeutic potential has been hampered.
Several additional factors resulting from the well-regulated biological roles of the various HDAC isoforms, impose further challenges for the conventional approaches to the design of HDACi. Experiments have shown that the amount of acetylated histones increases in the presence of HDACi. Yet, recruitment of HATs and HDACs by DNA-bound transcription factors results in the formation of multi-protein transcription regulatory complexes that confer cell type specificity and signal dependent regulation to arrays of subordinate genes. Using HDACi that inhibit HDACs indiscriminately is akin to throwing a monkey ranch into a complicated and delicately balanced machine. This explains the numerous undesirable side-effects observed in many of the trials involving HDACi.
The problems encountered in investigating medicinal uses of HDACi are also shared by researchers investigating epigenetic regulation. Epigenetic regulation is the establishment of inheritable gene expression patterns without permanently changing the DNA sequence. It has emerged as a key mechanism for regulating cellular function.
Alteration of epigenetic regulation is a hallmark of many diseases, especially cancer. Small molecules that are being developed as drugs in treating these diseases, typically found via phenotypic screening, act by modulating the epigenetic control of cellular process. As such, the fundamental mechanisms of epigenetic regulation is an area of intense interest. At the same time, the search for small molecule epigenetic regulators is becoming a very promising area for drug discovery.
Because epigenetic regulation is achieved largely through chemical modifications of chromatin structure by enzymes that act upon DNA (e.g. DNA cytosine methyltransferase) or proteins (e.g. HATs, HDACs, histone methylases, and histone demethylases), in this context, HDACs' role in regulating DNA transcription can be viewed as a component of epigenetic regulation. Similar to HDACi, the majority of current chemical modulators of epigenetic regulators inhibit these enzymes by binding to their catalytic site, which is often shared by multiple enzymes with distinct cellular roles.
Despite recent advances regarding the role of transcription factors such as MEF2, and its implication in several major diseases, it has not been possible to identify small molecules that are capable of modulating the function of MEF2. Such molecules would facilitate further advances in this field and can lead to new mechanism-based and structure-based therapeutic applications for MEF2-associated diseases, including inflammation, autoimmune diseases, neurodegenerative diseases, cancer, and cardiovascular disease.
Therefore, a general problem in this area of research has been the lack of small molecules that can target a specific epigenetic regulator enzyme or protein. A further problem has also been the lack of methods for the design, evaluation and optimization of such small molecules, in a manner that bestows the required selectivity without the associated drawbacks resulting from broad-spectrum activities across entire enzyme or protein classes. Such molecules will have important applications as molecular tools in studying the basic mechanism of epigenetic regulation as well as therapeutic agents for targeted therapeutic interventions.